Arcobacteraceae: An Exploration of Antibiotic Resistance Featuring the Latest Research Updates

The Arcobacteraceae bacterial family includes species isolated from animals and related food products. Moreover, these species have been found in other ecological niches, including water. Some species, particularly Arcobacter butzleri and Arcobacter cryaerophilus, have been isolated from human clinical cases and linked to gastrointestinal symptoms. The presence of antibiotic-resistant strains is a concern for public health, considering the possible zoonoses and foodborne infections caused by contaminated food containing bacteria resistant to antibiotic treatments. This review aims to highlight the importance of antibiotic resistance in Arcobacter spp. isolates from several sources, including information about antibiotic classes to which this bacterium has shown resistance. Arcobacter spp. demonstrated a wide spectrum of antibiotic resistance, including several antibiotic resistance genes. Antibiotic resistance genomic traits include efflux pumps and mutations in antibiotic target proteins. The literature shows a high proportion of Arcobacter spp. that are multidrug-resistant. However, studies in the literature have primarily focused on the evaluation of antibiotic resistance in A. butzleri and A. cryaerophilus, as these species are frequently isolated from various sources. These aspects underline the necessity of studies focused on several Arcobacter species that could potentially be isolated from several sources.


Introduction
The Arcobacteraceae bacterial family includes Gram-negative species isolated from several environment matrices and hosts.Some of these species have been isolated from animals in which these bacteria have shown pathogenicity.Recently, a division between pathogenic and non-pathogenic strains has been proposed [1].Arcobacter butzleri and Arcobacter cryaerophilus are considered the two species in the Arcobacteraceae family that are most frequently associated with clinical outbreaks.Although to a lesser extent, Arcobacter cibarius, Arcobacter thereius, and Arcobacter skirrowii are also considered pathogens.The main symptoms of Arcobacter spp.infection are related to gastrointestinal disorders, with diarrhoea being the most prominent.Arcobacter spp. is widely considered a zoonotic pathogen related to foodborne diseases.Furthermore, it is important to consider that Arcobacter spp.can be mistaken for Campylobacter spp.during clinical analyses, warranting additional attention to this pathogen [2].The species included in the Arcobacteraceae family usually do not cause symptoms in animals [3].The asymptomatic behaviour of these bacteria can increase their spread, making them more difficult to identify directly.Although Arcobacter infection often remains asymptomatic, these bacteria have been associated with various symptoms in some cases.A. butzleri has been linked to enteritis, with symptoms of diarrhoea in cattle, pigs, and horses [4,5].A. butzleri has also been isolated from faecal samples of chickens, turkeys, ducks, and domestic geese [6,7].In research performed in Türkiye, A. butzleri was the species most frequently isolated from chickens, geese, ducks, turkeys, and quails, followed by A. cryaerophilus, A. skirrowii, and A. cibarius [8].The species A. thereius has been isolated from pigs and ducks in Belgium [9]. A. skirrowii has been associated with diarrhoea and haemorrhagic colitis in cattle and sheep [5,10].
The isolation of Arcobacter spp.from animals can be linked to its presence in food [11].Food is considered one of the main transmission sources of Arcobacter spp., which, due to their pathogenicity, are considered foodborne pathogens.The principal foods found to be contaminated by Arcobacter spp.are of animal origins (clams, milk, meat, and fish), with chicken meat showing a high percentage of isolation related to this bacterial genus.However, Arcobacter spp.have also been found in vegetables and ready-to-eat vegetables.Regarding vegetables, Arcobacter spp.have been detected on lettuce [12], rocket [13], napa cabbage, water parsley [14], and ready-to-eat salad [15].The species most frequently isolated from vegetables is A. butzleri, while A. cryaerophilus has been isolated from leafy green vegetables [14].A. butzleri can survive in the apple and pear puree production process, although with a significant reduction in the bacterial load [16].
Considering the isolation of Arcobacter spp.from clinical cases and foods, and its pathogenicity in vitro, antibiotic-resistant strains represent a risk to public health.This aspect is related to the loss of antibiotic efficacy in case of infections [17].Moreover, the possible horizontal gene transfer of antibiotic resistance genes to other bacteria cannot be excluded.Arcobacter spp.represents a widely distributed human pathogen among foods, water, animals, and environmental niches [1].The antimicrobial resistance (AMR) of Arcobacter spp.underlines its importance as a pathogen due to the possible risk of infection after contact with contaminated sources treatable with reduced effectiveness due to decreased or absent effects of antibiotics.This review will discuss the AMR of Arcobacter spp., highlighting observations related to antibiotic-resistant strains from different sources, including food, water, and animals.Information about the mechanisms of Arcobacter spp. of antibiotic resistance mechanisms will also be discussed.This review aims to highlight the antibiotic resistance of Arcobacter spp., focusing on pathogenic species in humans with data from recent studies.

Antibiotic Resistance of Arcobacter Spp. Isolated from Food and Related Land Animals
Arcobacter spp.strains, isolated from foods, are resistant to several antibiotic classes (Table 1).The AMR of Arcobacter spp.has been demonstrated for different species isolated from various sources.Specific protocols for the AMR resistance evaluation of Arcobacter spp.are not available in the official guidelines [18].For this reason, breakpoint and method references will be indicated in the text considering the different methods used in the AMR determination of Arcobacter spp.These specifications are rendered necessary because different AMR evaluation methods may lead to different results [19,20].In Portugal, Arcobacter spp.showed multidrug resistance (MDR) following the antibiotic dilution method (European Committee on Antimicrobial Susceptibility Testing; EUCAST breakpoints) in 85.7% of the isolates from food samples [21].These authors demonstrated a high AMR in A. butzleri and A. cryaerophilus, especially to nalidixic acid (100% of A. butzleri and 87.5% of A. cryaerophilus isolates), tetracycline (95.4% of A. butzleri and 93.8% of A. cryaerophilus isolates), and cefotaxime (98.5% of A. butzleri and 93.8% of A. cryaerophilus isolates), while gentamicin was effective against all isolates [21].High resistance of A. butzleri to nalidixic acid (agar dilution method; Centers for Disease Control and Prevention; CDS and Clinical & Laboratory Standards Institute; CLSI break points) was confirmed by Isidro and colleagues in 22 strains from poultry samples, meats and vegetables, raw milks, and from a dairy plant environment, resulting instead in susceptibility to gentamicin [22].Resistance to cefoperazone (disk diffusion method; CLSI breakpoints) has been demonstrated for A. butzleri, A. cryaerophilus, and A. skirrowii isolated from meat [23].The evaluation of AMR in Arcobacter spp.from several sources (poultry meat, patients, and water) following a disk diffusion method (CLSI breakpoints) shows that most isolates were resistant to β-lactam antibiotics [24].In the case of poultry meat, A. butzleri was found to be antibiotic-resistant to ampicillin, aztreonam, cephalothin, clindamycin, nalidixic acid, oxacillin, and penicillin G, while A. cryaerophilus isolates were resistant to clindamycin, oxacillin, and penicillin G [24].Ampicillin, erythromycin, and tetracycline showed low efficacy against A. butzleri from chicken and cattle meat after a disk diffusion method evaluation (EUCAST breakpoints) [25].A. skirrowii isolated from poultry water was found to be resistant to streptomycin following a gradient strip diffusion method (E-test; EUCAST breakpoints) [26]. A. butzleri, A. cryaerophilus, and A. skirrowii from chicken samples in Egypt showed resistance (disk diffusion method; CLSI breakpoints) against ampicillin, ampicillin-sulbactam, and cefotaxime [27].Although the literature primarily focused on isolates from poultry meat, cases of AMR have been observed for Arcobacter spp.isolated from other meats.A high number of isolates resistant to cefotaxime, nalidixic acid, and tetracycline was observed for Arcobacter spp.isolates from pork and beef meat (antibiotic dilutions method; EUCAST breakpoints) [21].A study on A. butzleri from fresh raw cattle meat samples showed AMR (disk diffusion method; CLSI breakpoints) to tetracycline (72%), amoxicillin (69%), erythromycin (67%), and cefoxitin (66%), while 60% of A. cryaerophilus isolates were resistant to cefoxitin and erythromycin, confirming MDR phenomena in these species [28].Other important foods of animal origin in which Arcobacter spp.has been isolated are milk and dairy products.A study conducted in Iran demonstrated resistance to amoxicillin-clavulanic acid and tetracycline in A. butzleri isolated from milk, with some cases of AMR (disk diffusion method) for A. cryaerophilus isolates from the same matrix [29].A. butzleri and A. cryaerophilus isolated from milk were found to be resistant to amoxicillinclavulanic acid and tetracycline [29].Strains of A. butzleri isolated from chicken breast and fresh vegetables demonstrate MDR (disk diffusion method; EUCAST breakpoints) to tetracyclines and cefotaxime (third-generation antibiotic) [30].
Arcobacter spp.have been isolated from several land animals, including farm animals.However, most research on AMR in Arcobacter spp.has been focused on animal products such as milk and meat; for this reason, only a few recent works are mentioned here.A. butzleri isolates from healthy pigs' faecal samples (n = 203) showed resistance (disk diffusion method, CLSI) to cefotaxime in 98.6% of isolates, and 71% of isolates showed resistance to sulbactam-cefoperazone followed by ampicillin (67.7%), while AMR to enrofloxacin (48.4%) and fosfomycin (42.9%) was lower [31].Arcobacter spp., with a prevalence of A. butzleri, isolated from pigs, ducks, quails, and sheep in Ghana and Tanzania showed a 100% antibiotic resistance rate to ampicillin, chloramphenicol, and penicillin (disk diffusion method, EUCAST) [32].
The ability of Arcobacter spp. to colonize several surfaces has also been demonstrated [33,34].Some recent studies on Arcobacter spp.isolated from food processing plant surfaces are present in the literature.A. butzleri isolated from a dairy plant in Portugal showed resistance to nalidixic acid and susceptibility to erythromycin and gentamicin [31].However, isolates from slaughterhouse surfaces, even when showing resistance to ampicillin and nalidixic acid, also demonstrated resistance to erythromycin, indicating variable results between isolates from different sources [35].A. butzleri strains from a chicken slaughterhouse in Italy (chicken skins, cloacae, and surfaces) [36] demonstrated MDR (agar diffusion method, EUCAST breakpoints) to amoxicillin-clavulanic acid, amoxicillin, ampicillin, azithromycin, clarithromycin, erythromycin, and gentamicin [37].
The wide prevalence of antibiotic-resistant Arcobacter spp.strains in food and production plants, in addition to their pathogenic potential, underlines their dangers as food contaminants.This is even more evident considering that antibiotic resistance leads to a loss of antibiotic efficacy, resulting in difficulties in treating bacterial infections [16].
As stated, Arcobacter spp.has been isolated from water animals and related food products.AMR tests were performed on these isolates.Strains of A. butzleri isolated from sushi showed MDR (disk diffusion method, EUCAST) to tetracyclines and cefotaxime [30].A study conducted in Italy showed the presence of AMR Arcobacter strains in mussels and clams from a local fish market (disk diffusion method, CLSI) [42].Two strains showed high resistance to β-lactams (ampicillin, penicillin, and cefotaxime) as well as tetracycline, and erythromycin [42].Other authors demonstrated a high AMR (disk diffusion method, CLSI) of A. butzleri isolated from seafood to cephalothin, cefoxitin, and sulfamethizole [43].Arcobacter spp. was isolated from catla (Catla catla) samples from markets and aquaculture ponds, demonstrating MDR (disk diffusion method, CLSI) in five isolates of A. butzleri [44].Three of these isolates showed resistance to penicillin and cefixime, while two isolates showed resistance to penicillin, nalidixic acid, and erythromycin [44]. A. butzleri strains from clams (Tapes philippinarumand) and mussels (Mytilus galloprovincialis) were found to be resistant to ampicillin, penicillin, cefotaxime, tetracycline, and erythromycin, while one strain was resistant to nalidixic acid (disk diffusion method, CLSI) [42].
The widespread presence of Arcobacter spp. in water and water animals and their AMR draws attention to the risk associated with ingesting antimicrobial-resistant strains from these sources.

Antibiotic Resistance of Arcobacter Spp. Isolated from Humans
Species of Arcobacter spp., prevalently A. butzleri and A. cryaerophilus, have been isolated from human clinical cases (Table 3).Clinical cases related to Arcobacter spp.are normally solved without the need for antibiotic treatment [45].However, in some cases, treatment has been necessary.A study that included samples from German patients from whom A. butzleri, A. cryaerophilus, and Arcobacter lanthieri had been isolated demonstrated that ciprofloxacin (E-test; CLSI) was the most appropriate antibiotic among those tested [46].An Arcobacter spp.infection in a COVID-19 and HIV patient was resolved with a treatment that included intravenous meropenem for five days followed by oral ciprofloxacin [47]. A. lanthieri was isolated in Belgium from a patient with abdominal bloating and cramps [48].In this case, the infection resolved spontaneously, but the isolate showed AMR (E-test; EUCAST) to ampicillin, ciprofloxacin, and erythromycin [48].
The in vitro AMR of Arcobacter spp.isolated from clinical samples has been observed.A. butzleri and A. cryaerophilus isolated from Belgian patients were found to be resistant (E-test; EUCAST) to ampicillin (91% of the strains) [49].A study conducted in A. butzleri isolates from clinical samples showed high AMR (E-test; CLSI) to ampicillin (MIC; 24-64 µg/mL) [46].Two A. butzleri strains isolated from a patient with travellers' diarrhoea and from another with pruritus showed AMR to tetracycline, while amoxicillin-clavulanic acid and ampicillin AMRs (MIC test strip; EUCAST) were observed in one strain [50].A study performed in Central Italy demonstrated AMR in an A. butzleri strain to amoxicillinclavulanic acid, ampicillin, tetracycline, ciprofloxacin, nalidixic acid, cefalotin, cefotaxime, erythromycin, gentamicin, and streptomycin (disk diffusion test; EUCAST and CLSI) [30].Another strain from the same study was susceptible to amoxicillin-clavulanic acid and showed intermediate resistance to gentamicin [30].Šilha and colleagues observed a high AMR ratio in A. butzleri from human enteritis cases, with at least six of the seven strains tested resistant to ampicillin, aztreonam, chloramphenicol, clindamycin, nalidixic acid, oxacillin, and penicillin G (disk diffusion test; CLSI) [24].All A. butzleri, A. cryaerophilus, and A. skirrowii isolated in a study conducted in Iran demonstrated AMR against cefazolin, ceftazidime, and nalidixic acid (disk diffusion test; CLSI) [51].Moreover, all A. butzleri isolates demonstrated AMR to chloramphenicol [51].
The AMR assays demonstrate that Arcobacter spp.show resistance to several antibiotic classes even in isolates from human clinical cases.This aspect underlines the importance of Arcobacter spp. as a bacterial pathogen.

Genomic Traits Related to Antibiotic Resistance
The high AMR of Arcobacter spp.suggests the presence of genomic determinants in its genome (Figure 1).The antibiotic resistance of Arcobacter spp.has been correlated to specific genetic factors.Isidro and colleagues linked the AMR (agar dilutions method; CLSI breakpoints) of A. butzleri to fluoroquinolones with Thr-85-Ile in GyrA, while ampicillin resistance was associated to OXA-15-like β-lactamase [22].Similarly, A. cryaerophilus isolated from water poultry and resistant (E-test; EUCAST breakpoints) to ciprofloxacin showed a point mutation (Thr-85-Ile) in gyrA [52]. A. butzleri and A. cryaerophilus isolated from water sources presented tetW (tetracycline resistance), while A. butzleri was also characterized by tetO and tetA [40].A study conducted on antibiotic-resistant A. butzleri isolates (disk diffusion method; CLSI breakpoint) from shellfish determined the presence of DegT/DnrJ/EryC1/StrS aminotransferase family protein, which is required for the resistance to polymyxin and cationic antimicrobial peptides and HipA (type II toxinantitoxin system) involved in methicillin resistance [42].The same authors detected the presence of outer membrane efflux protein-related genes linked to AMR; among these were the genes feoA and feoB [42].Antibiotic resistance genes blaOXA-61, tetO, and tetW were found in all A. butzleri, A. cryaerophilus, and A. lacus isolates obtained from seafood and water samples [53].Colistin resistance genes (mcr1/2/6, mcr3/7, mcr4, mcr5, and mcr8) where found in part of the isolates, with mcr5 present in all A. cryaerophilus isolates [53].
A study conducted in China demonstrated that A. butzleri and A. cryaerophilus isolated from pork and chicken harboured resistance island gene clusters, while an A. butzleri isolate showed ereA, a macrolide resistance gene [54].A. butzleri and A. cryaerophilus isolated from cattle meat demonstrated the presence of the AMR genes qnr (quinolone resistance gene), dfrA1 (dihydrofolate reductase), tetB and tetA (tetracycline resistance), blaCITM and blaSHV (beta-lactam resistance), and sul1 (sulfonamide resistance) [28].Genomes of A. butzleri isolates from human clinical cases contained tetO, linked to tetracycline resistance, and bla3, linked to ampicillin and amoxicillin-clavulanic resistance [50].The presence of AMR genes revealed its influence on the antibiotic resistance of Arcobacter to several antibiotics.Strains of A. butzleri isolated from cow milk harboured the adeF gene (present in all strains, conferring resistance to fluoroquinolone and tetracycline), while 90% of the strains harboured the acrB gene (conferring resistance to rifamycin, cephalosporin, triclosan, glycylcycline, tetracycline, penam, phenicol, and fluoroquinolone) [55].Some 30% of strains demonstrated the presence of pmrE (conferring resistance to polypeptide antibiotics), while 10% of strains carried aadA2 (aminoglycoside resistance) and macB (macrolide resistance) [55].Additionally, in this work, the mutations S140N, A139V, R463L, and A379T of the katG gene, conferring resistance to isoniazid, were detected in 50% of the strains [55].
Similarly to the mentioned gyrA, genetic variants and orthologues can differentially influence antibiotic resistance.A study conducted on 31 A. butzleri strains isolated from chicken carcasses and slaughterhouse equipment demonstrated a correlation between hlyD orthologues and AMR to several antibiotics (agar diffusion method, EUCAST breakpoints) [37].The same pangenome study demonstrated a correlation of RND efflux pump and hydrophobe/amphiphile efflux-1 with AMR and a correlation of mexAB-oprM operon and cydB with MDR [37].Another study on A. butzleri isolates from poultry suggested the importance of oxa-464 and T81I point mutations in the quinolone resistance-determining region (disk diffusion method; EUCAST and CLSI breakpoints) [56].

Conclusions
Arcobacter spp. is considered an emergent foodborne pathogen, characterized by high persistence in food production plants [37].Moreover, Arcobacter spp.'s presence in animals is well known [3].For these reasons, the emergence of resistance to several antibiotic classes is considered an additional public health risk due to clinical treatment ineffectiveness (Figure 1, Tables 1-3) [58].As stated, recent studies highlighted the MDR of Arcobacter, spp.including to several classes.Arcobacter spp.demonstrated a wide range of AMR traits (Figure 1).This can be linked to the presence of efflux pumps that can confer AMR to a wide range of antibiotics and to specific AMR genes.However, the high presence of hypothetical proteins in Arcobacter spp.[1] limits a comprehensive genome exploration linked to AMR.Even if procedures recommended by the Clinical and Laboratory Standards Institute and the European Committee on Antimicrobial Susceptibility Testing for Campylobacter or Enterobacterales are normally used for Arcobacter spp., the absence of standard procedures in AMR determination [18] can lead to different results between authors.This suggests the necessity of including Arcobacter spp. in official internationally recognized procedures.The current knowledge about Arcobacter spp.AMR is principally focused on A. butzleri, followed by A. cryaerophilus.Moreover, the number of studies focused on clinical isolates is low compared to food-related studies.Further studies are needed to increase the knowledge about AMR in this bacterial genus, including additional species and isolation sources.Moreover, an approach based on genomic analysis to be correlated to in vitro antibiotic studies and gene transformation of possible candidate resistance genes will allow for more precise identification of genetic traits linked to antibiotic resistance.This will enable the design of new analytical methods for the detection of Arcobacter spp.resistant to antibiotics.

Figure 1 .
Figure 1.AMR mechanisms in Arcobacter spp.The figure shows genomic traits at which Arcobacter spp.resulted in antibiotic resistance or that were detected through molecular methods.Antibiotics/classes and related mechanisms of action are included in the red and green boxes.The protein figures were uploaded from Uniprot (https://www.uniprot.org/;accessed on 7 June 2024) [57].

Table 1 .
Species of Arcobacter spp.showing AMR to several antibiotic classes, isolated from meat, food, and related animals.The table indicates antibiotics, their classes, and the sources from which Arcobacter spp.showed resistance.

Table 2 .
AMR of A. butzleri isolated from water, water environments, and related animals and food.The table indicates antibiotics, their classes, and the sources of isolation from which A. butzleri showed resistance.

Table 3 .
Species of Arcobacter spp.showing AMR to several antibiotic classes, isolated from clinical cases.The table indicates antibiotics, their classes, and the sources from which Arcobacter spp.showed resistance.Literature references are included in the last columns.